Registration Dossier

Toxicological information

Repeated dose toxicity: inhalation

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01-OCT-2002 to 29-MAY-2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: liquefied gas
Details on test material:
- Name of test material (as cited in study report): Hexafluoro-1,3-butadiene

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Crl:CD (SD) IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Uk) Ltd / Margate, Kent / ENGLAND
- Age at study initiation: 55 to 62 days of age
- Weight at study initiation: 271 to 320 g for males; 207 to 260 g for females
- Fasting period before study: no data available
- Housing: inside a rodent facility with a positive pressure, five of one sex per stainless steel cage (North Kent Plastic; 35 cm x 53 cm x 25 cm)
- Diet: ad libitum, except during the 6-h exposure or when urine was being collected and overnight before routine blood sampling; Rat and Mouse No. 1 Maintenance Diet (Special Diet Services / Witham / ENGLAND)
- Water: ad libitum, except during the 6-h exposure or when urine was being collected; community tap-water
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 22.5°C
- Humidity: 31 to 52%
- Air changes: no data available
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From 09-OCT-2002 to 19-NOV-2002 (main study) and 03-DEC-2002 (recovery study)

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: clean air
Remarks on MMAD:
MMAD / GSD: Not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: whole-body exposure chambers constructed from stainless steel and glass, with an internal volume of 0.75 cm3
- Source and rate of air, method of conditioning air: no data available
- System of generating atmosphere: the test atmosphere was produced by metering the test liquid from a stainless steel pressure resistant cylinder, followed by dilution with clean air prior to the resultant vapour atmosphere passing into the exposure chamber.
- Temperature, humidity, pressure in air chamber, air flow rate, air change rate, treatment of exhaust air: no data available

TEST ATMOSPHERE
No data available

VEHICLE
No data available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No data available
Duration of treatment / exposure:
4 consecutive weeks
Frequency of treatment:
6 hours/day
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
5 ppm (nominal)
Remarks:
corresponding to 5 ppm (analytical) or 0.033 mg/L (converted) or 33 mg/m3 (converted)
Dose / conc.:
15 ppm (nominal)
Remarks:
corresponding to 15 ppm (analytical) or 0.099 mg/L (converted) or 99 mg/m3 (converted)
Dose / conc.:
50 ppm (nominal)
Remarks:
corresponding to 51 ppm (analytical) or 0.338 mg/L (converted) or 338 mg/m3 (converted)
No. of animals per sex per dose:
5 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the target exposure levels used in this study were selected based on a previous work with this compound performed in the laboratory (see ESR entitled "SIFREN - Repeat. dose tox.: 14-day inhal. - K1 2003HLS"). In this study, rats were exposed to target concentrations of 10, 30 and 100 ppm, where clinical effects were seen at the higher exposure levels.
- Rationale for selecting satellite groups, post-exposure recovery period in satellite groups: in order to assess the reversibility of any treatment-related findings satellite groups of animals were maintained for a subsequent period of 2 weeks without treatment. Male and female rats (5/sex/dose) were exposed either to clean air (control) or 50 ppm of the test item daily for 4 weeks followed by the 2-week recovery period.

Examinations

Observations and examinations performed and frequency:
Debilitated animals were observed carefully and, where necessary, isolated to prevent cannibalism. Animals judged in extremis were killed. Animals were also killed to prevent unnecessary or prolonged suffering. Where possible, blood samples were taken ante mortem and analysed for haematology and blood chemistry parameters. A complete necropsy was performed in all cases.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule, cage side observations checked: animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the occupants, such as loose faeces.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed observations were recorded daily, on the days of exposure as follows: pre-exposure observation, during exposure, as each animal was returned to its home cage, and as late as possible in the working day. In addition, a more detailed weekly physical examination was performed on each animal to monitor general health. During the acclimatisation and recovery periods, observations of animals and their cages were recorded at least once per day.
Before treatment and during each week of treatment and week 2 of recovery period, a detailed physical examination and arena observations were performed on each animal. Examinations were performed at approximately the same time of day.
After removal from home cage, animals were assessed for physical condition and behaviour during handling and after being placed in standard arena. Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremors and abnormalities of gait or behaviour. Attention was also given to the detection of audible respiratory noise.

BODY WEIGHT: Yes
- Time schedule for examinations: the weight of each rat was recorded 1 week prior to the start of exposure (week -1), on the day that treatment started (day 0), weekly throughout the exposure and recovery periods, and before necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; the weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded 1 week prior to the start of exposure (week -1), and weekly throughout the exposure and recovery periods. From these records the mean weekly consumption per animal (g/animal) was calculated for each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: the quantity of water consumed by each cage of rats was recorded daily, commencing 1 week prior to the start of exposures and throughout the exposure and recovery periods.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the start of exposures and during week 4 of treatment
- Dose groups that were examined: all animals before the beginning of exposures and animals from groups 1 (control) and 4 (high dose) during week 4 of treatment
As no treatment-related changes were observed, the examination was not performed during the recovery phase or for animals of groups 2 and 3.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to sacrifice following the last day of exposure and the last day of the recovery period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes; overnight starvation
- How many animals: all animals of control and treated groups
- Parameters checked: haematocrit (Hct); haemoglobin (Hb); red blood cell count (RBC); mean cell haemoglobin (MCH); mean cell haemoglobin concentration (MCHC); mean cell volume (MCV); total white cell count (WBC); differential WBC count (neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M), large unstained cells (LUC)); platelet count (Plt); reticulocyte count (Retic); blood cell morphology/type; prothrombin time (PT); activated partial thromboplastin time (APPT); cellularity (cellular), distribution (Distrib) of cell types and cell morphology (Morp) of tibial bone marrow samples

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to sacrifice following the last day of exposure and the last day of the recovery period
- Animals fasted: Yes; overnight starvation
- How many animals: all animals of control and treated groups
- Parameters checked: alkaline phosphatase (ALP); alanine aminotransferase (ALT); aspartate aminotransferase (AST); gamma-glutamyl transpeptidase (gGT); creatine phosphokinase (CPK total); total bilirubin (Bili); urea; creatinine (Creat); glucose (Gluc); total cholesterol (Chol); triglylcerides (Trig); sodium (Na); potassium (K); chloride (Cl); calcium (Ca); inorganic phosphorus (Phos); Total protein (total Prot); albumin (Alb); α1 globulin (a1); α2 globulin (a2); β globulin (Beta); γ globulin (Gamma); albumin/globulin ratio (A/G ratio)

URINALYSIS: Yes
- Time schedule for collection of urine: during week 4 of treatment and week 2 of recovery
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes; from approximately 17:40 hours (week 4 of treatment) and 16:30 hours (week 2 of recovery) until approximately 07:30 hours the following day
- Parameters checked: appearance (App); volume (Vol); pH; specific gravity (SG); protein (Prot); sodium (U-Na); potassium (U-K); chloride (U-Cl); glucose (Gluc); ketones (Keto); bile pigments (Bili); heam pigments (Blood); epithelial cells (Epi); leucocytes (Leuc); erythrocytes (RBC); crystals (Cryst); spermatozoa and precursors (Sperm); casts (Casts); other abnormal components (Abn.)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4 of treatment, as well as during week 2 of recovery.
- Dose groups that were examined: all animals of control and treated groups were investigated. However, animals were not necessarily all tested on the same day, but the number of animals was balanced across the groups on each day of testing.
- Battery of functions tested: sensory activity (i.e., approach response, touch response, auditory startle reflex, tail pinch response), grip strength (i.e., forelimb and hindlimb), motor activity

OTHER:
* URINARY FLUORIDE: following assessment of the above urinary parameters, the residual urine samples were kept at approximately -20°C for further analysis of urinary fluoride by ion specific electrode.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; animals killed during the study and those surviving until the end of the scheduled treatment or recovery period were killed by an intraperitoneal injection of pentobarbitone sodium followed by exsanguination from the brachial arteries. All animals were then subjected to a detailed necropsy. A full macroscopic examination of tissues was performed. The cranial roof was removed to allow observation of brain, pituitary gland and cranial nerves. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. The requisite organs were weighed and external and cut surfaces of organs and tissues were examined. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

HISTOPATHOLOGY: Yes; testes and epididymides were fixed in Bouin's solution and eyes were fixed in Davidson's fluid, prior to transfer of these tissues to 70% industrial methylated spirit. Samples (or the whole) of the other tissues listed below from main study and recovery animals were preserved in 10% neutral buffered formalin, dehydrated, embedded in paraffin wax, sectioned at 4- to 5-µm thickness and stained with hematoxylin and eosin, except the testes which were stained using a standard periodic acid/Schiff (PAS) method:
Adrenals - cortex and medulla
Aorta - thoracic
Brain - cerebellum, cerebrum and midbrain
Caecum
Colon
Duodenum
Epididymides
Eyes
Femur with joint - longitudinal section through the joint
Harderian glands
Head (not examined histologically but retained against any requirement for microscopic examination)
Heart - including cortex, medulla and papilla regions
Ileum
Jejunum
Kidneys
Lachrymal glands
Larynx - 2 levels
Liver - section from all major lobes
Lungs - section from all major lobes, including bronchi
Lymph nodes - mandibular, mesenteric, tracheo-bronchial
Mammary area - caudal (not examined histologically but retained against any requirement for microscopic examination)
Nasal turbinates - 3 levels
Nasopharynx
Oesophagus
Optic nerves (not examined histologically but retained against any requirement for microscopic examination)
Ovaries
Pancreas
Pituitary
Prostate (not examined histologically but retained against any requirement for microscopic examination)
Rectum
Salivary glands - submandibular/sublingual
Sciatic nerves
Seminal vesicles
Skeletal muscle - thigh
Skin (not examined histologically but retained against any requirement for microscopic examination)
Spinal cord - transverse and longitudinal section at the cervical, lumbar and thoracic levels
Spleen
Sternum - including bone marrow
Stomach - including keratinised, glandular and antrum in sections
Testes
Thymus
Thyroid - including parathyroids in section where possible
Tongue (not examined histologically but retained against any requirement for microscopic examination)
Trachea - including bifurcation
Urinary bladder
Ureter (not examined histologically but retained against any requirement for microscopic examination)
Uterus and cervix
Vagina (not examined histologically but retained against any requirement for microscopic examination)
For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required for microscopic pathology.
Samples of any abnormal tissues were also retained and processed for examination. In those cases where a lesion was not clearly delineated, contiguous tissue was fixed with the grossly affected region and sectioned as appropriate.

Microscopic examination was performed as follows: all tissues preserved for examination were investigated for all main study animals of groups 1 (control) and 4 (high dose) sacrificed on completion of the scheduled treatment period, and for all animals killed or dying during the study. Microscopic examinations for all main animals of groups 2 and 3 and all recovery animals was limited to adrenals, epididymides, femur, heart, kidneys, liver, lungs, spleen and testes.
Tissues reported at macroscopic examination as being grossly abnormal were examined for all animals of main and recovery studies.
In addition, the following tissues, considered to exhibit a reaction to treatment at the high exposure level, were examined for all animals of groups 2 and 3 in the main study and in all animals of recovery study: pancreas (males only) and nasal turbinates.
Other examinations:
ORGAN WEIGHTS: the following organs, taken from each animal killed after 4 weeks of treatment or 2 weeks of recovery, were dissected free of adjacent fat and other contiguous tissue and the weighs were recorded:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Lungs including bronchi
Ovaries
Spleen
Testes
Thymus
Uterus with cervix
Bilateral organs were weighed together.
Statistics:
All statistical analyses were carried out separately for males and females.
The following data types were analysed at each time point separately:
- Grip strength and motor activity
- Bodyweight (using gains)
- Blood chemistry, haematology and urinalysis (including urinary fluoride)
- Organ weights (absolute and adjusted for terminal bodyweight)
- Pathological findings (for the number of animals with and without each finding)
For categorical data, including pathological findings, the proportion of animals was analysed using Fisher's exact test for each treated group vs. the control.
For continuous data, Bartlett's test was first applied to test the homogeneity of variance between groups. Using tests dependent on the outcome of Bartlett's test, treated groups were then compared with the control group, incorporating adjustment for multiple comparisons where necessary.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
there were no clinical signs observed at any time prior to exposure, during exposure, post-exposure or during the weekly physical examination and arena observations.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
there was one unscheduled death.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
see in "Details on results"
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
see in "Details on results"
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
see in "Details on results"
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
see in "Details on results"
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
see in "Details on results"
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
see in "Details on results"
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
see in "Details on results"
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
One female of group 4 was sacrificed following exposure on day 14 of the main study due to suspected broken bone. There were no clinical signs observed at any time prior to exposure, during exposure, post- exposure or during the weekly physical examination and arena observations. Brown staining on head and red/brown staining around eyes were noted for a small proportion of rats on a number of occasions following exposure.

BODY WEIGHT AND WEIGHT GAIN (see Table 3 in "Any other information on results incl. tables")
The loss in bodyweight seen for some groups at week 4 of exposure period was considered to be caused by overnight deprivation of food, on day 28, prior to bleeding for haematology and blood chemistry investigations.
Examination of weight gain up to week 3 of exposure period indicated a significant decrease in weigh t gain for females of group 4 when compared to controls. During the recovery period, bodyweights at recovery week 2 were again affected by overnight starvation. At week 1 of recovery, bodyweight gains of groups 1 and 4 were similar.


FOOD AND WATER CONSUMPTION (see Table 3 in "Any other information on results incl. tables")
The reduction in food and water consumption observed during week 4 of exposure period and week 2 of recovery period was considered attributable to the overnight deprivation of food, on day 28 of exposure period and day 14 of recovery period, respectively, prior to bleeding for haematology and blood chemistry investigations and was considered not to be treatment-related.
Examination of food and water consumption up to week 3 of exposure period and at week 1 of recovery indicated no statistically significant change in all treated groups.

OPHTHALMOSCOPIC EXAMINATION
There were no significant abnormalities observed during the pre-treatment ophthalmic examination, as well as during the examination at week 4 of exposure period.

HAEMATOLOGY
In peripheral blood, differences between groups were generally small, independent of dosage and largely inconsistent between sexes. They were considered not to be of toxicological importance. Visual assessment of bone marrow smears for cellularity, distribution and morphology indicated that there were no treatment-related effects on bone marrow.

CLINICAL CHEMISTRY (see Table 4 in "Any other information on results incl. tables")
Blood urea levels were higher than control in all treated groups following 4 weeks of exposure, however, this was not dose-related. Statistical significance was achieved for male rats of groups 3 and 4 and female rats of group 4. At the end of recovery period, urea levels were similar for groups 1 and 4.

Other differences were generally small, independent of dosage and largely inconsistent between the sexes. They were considered unlikely to be of toxicological importance.

URINALYSIS (see Table 5 in "Any other information on results incl. tables")
Group mean urine volume was lower than control for all treated groups and although the effect was greatest in group 4 there was no clear dose-relationship. None of the differences were statistically significant. The reduced urine volume in group 4 was associated with increased specific gravity, urinary K and Cl concentrations, which were statistically significant for females. The effect on urine volume and electrolyte concentration was not apparent after the 2-week recovery period and was therefore considered likely to be a treatment-related effect.
Other differences were generally small, independent of dosage and largely inconsistent between the sexes. They were considered unlikely to be of toxicological importance.

NEUROBEHAVIOUR (see Table 2 in "Any other information on results incl. tables")
There were no treatment-related effects on sensory reactivity and motor activity.
In contrast, a significant increase in hindlimb grip strength was evident for females of group 4 following 4 weeks of exposure. Moreover, a non-significant increase in forelimb grip strength was also evident for females of groups 3 and 4 following 4 weeks of exposure. A trend towards increased hindlimb grip strength was seen for males and females of group 4 following the 2-week recovery period; an upward trend in fore limb grip strength was also observed for females of group 4 at the end of recovery period. However, these increases were very slight.
The apparent increases in grip strength noted at the end of exposure and recovery periods were considered not to be treatment-related but rather to be due to natural variation. In addition, due to the isolated nature of the finding (i.e., in the absence of other sensory reactivity and motor activity treatment-related findings) and as the direction of the change (increase) for grip strength at the end of exposure period occurred while bodyweight was experiencing reduced gains, it was considered unlikely that the increase in grip strength was treatment-related.

ORGAN WEIGHTS (see Table 6 in "Any other information on results incl. tables")
Kidney weights (absolute and bodyweight adjusted values) for test groups were greater than control at week 4 of exposure period. Statistical analysis of the bodyweight adjusted values indicated that statistical significance was achieved for all female groups and males of group 4.
Liver weights (absolute and bodyweight adjusted values) were slightly higher than control values for all test groups but the effect was not dose-related, since weights of group 2 were higher than those of groups 3 and 4. In the absence of microscopic findings, it was considered that this increase in liver weight was of no toxicological importance. Statistical significance was achieved for bodyweight adjusted values for all female groups and males of group 4.
At the end of recovery period, kidney weights (absolute and bodyweight adjusted values) for females of group 4 remained higher than control.
All other statistically significant differences were considered not to be of toxicological significance.

GROSS PATHOLOGY
The macroscopic examination performed at termination revealed no lesions attributable to treatment. The incidence and distribution of all findings were considered to fall within the expected background range of macroscopic changes.

HISTOPATHOLOGY: NON-NEOPLASTIC (see Table 7 in "Any other information on results incl. tables") The following microscopic changes were considered to be treatment-related:
- Nasal turbinates: in the respiratory epithelium, an increased incidence of pseudogland formation was seen in males of group 4 compared to controls. Pseudogland formation was considered a minor response and was probably related to local goblet cell hyperplasia. After 2 weeks of recovery, the respiratory epithelium of nasal turbinates showed no more impairment, demonstrating a complete recovery.
- Kidneys: cortical tubules with hyaline droplets were noted in all male treated groups but not in any controls. After 2 weeks of recovery, only a single male in group 4 showed cortical tubules with hyaline droplets in the kidney.

In addition, an increased incidence and severity of acinar cell apoptosis were noted in males of group 4, compared with controls, but not in females of this group. As this finding was seen in controls of both sexes and the severity was not high compared to controls, its relationship to treatment was uncertain. After 2 weeks of recovery, no acinar cell apoptosis was seen in the pancreas of any animal, demonstrating a complete recovery.

No histological findings were observed in the liver to explain the increased organ weights recorded in high dose groups.

All other microscopic findings in animals from main and recovery studies were considered to be incidental and of no toxicological importance.

OTHER FINDINGS (see Table 5 in "Any other information on results incl. tables")
* URINARY FLUORIDE: a dose-related increase in urinary fluoride was evident in both sexes of all test groups following 4 weeks of exposure and attained statistical significance for groups 3 and 4. An increase in urinary fluoride was still evident for rats of group 4 following the 2-week recovery period. This was statistically significant but markedly less than that measured at week 4 of exposure period.

Effect levels

Key result
Dose descriptor:
NOAEC
Effect level:
5 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity

open allclose all
Critical effects observed:
yes
Lowest effective dose / conc.:
5 ppm (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
51 ppm (nominal)
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Dose response relationship:
yes

Any other information on results incl. tables

The pseudogland formation observed in the nasal turbinates of the nasal cavity of animals treated for 28 -day by inhalation was no longer present following the 2 week-recovery period.

The hyaline droplet formation in the cortical tubules of kidneys of male rats was reversed after the 2 week-recovery period (except 1 animal) and may be related to accumulation of alpha-2u globulin, an effect specific to male rats, and which is usually not considered relevant to humans.

The increased body weight-adjusted liver weight observed at the end of the 28-day treatment period was no longer significant after the 2-week recovery period and was considered adaptative in nature.

Applicant's summary and conclusion

Conclusions:
The only effects seen in rats exposed to 5 ppm of test item were a slight increase in hyaline droplet formation in the cortical tubules of kidneys (males only) and an increase in urinary fluoride (males and females). The former was considered to be of no significance to man and the latter was a consequence of the metabolic breakdown of the test substance.
At 15 and 51 ppm the adverse effects included microscopic changes in the nasal turbinates (statistically significant in males at 51 ppm) and a slightly reduced weight gain (statistically significant in females at 51 ppm). Based on these observations, the no-observed-adverse-effect-level, for male and female rats, was considered to be 5 ppm (eq. to 33 mg/m3).
Executive summary:

The objective of this study was to assess the systemic toxicity potential of the test substance to Crl:CD (SD)IGS BR rats by repeated inhalation administration over a period of 4 weeks, followed by a 2-week recovery period. The study was conducted according to OECD guideline 412 and in compliance with GLP.

Three groups of male and female rats (5/sex/dose) were exposed to a vapour of the test substance by inhalation at nominal concentrations of 5, 15, and 50 ppm, 6 hours a day for 4 consecutive weeks using a whole-body exposure system. A similarly constituted control group was exposed to clean air only. A further 5 male and 5 female rats were assigned to each of the control and high dose groups for a 2-week recovery period following the 4-week treatment period. During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, bodyweight, food and water consumption, ophthalmic examination, urinary fluoride, organ weight, macroscopic and microscopic pathology investigations were undertaken.

The study mean analysed concentrations of the test item were 5, 15 and 51 ppm for the low, intermediate and high dose groups, respectively. These concentrations were equivalent to 0.033, 0.099 and 0.338 mg/L (at 25°C and 760 mmHg) for groups 2 to 4, respectively.

There was one unscheduled death. A female of group 4 was sacrificed on humane grounds following exposure on day 14 due to a suspected broken bone. There were no treatment-related clinical signs observed pre-exposure, during exposure, post-exposure or during the weekly physical examination and arena observations.

There were no treatment-related effects on sensory reactivity, grip strength or motor activity.

Reduced mean bodyweight gains were evident for rats of groups 3 and 4 during the exposure period and attained statistical significance for female rats of group 4.

A slight reduction in food and water consumption was evident for females of group 3, and males (food only) and females (food, water) of group 4 during the exposure period. A slight reduction in food and water consumption was still evident for females of group 4 during the recovery period.

There were no significant abnormalities observed during the pre-treatment ophthalmic examination. There were no treatment-related effects noted during the examination in week 4.

There were no treatment-related effects on haematology parameters including those examined in the bone marrow.

Blood urea levels were higher than control in all treated groups following 4 weeks of exposure and attained statistical significance for male rats of groups 3 and 4, and females of group 4. At the end of recovery period, urea levels were similar for groups 1 and 4. The elevation of blood urea levels was considered likely to be a consequence of changes in renal function and was considered to be of no toxicological importance due to the lack of a dose-relationship.

Group mean urine volume was lower than control for all treated groups. The reduced urine volume in rats of group 4 was associated with increased specific gravity, urinary potassium concentration and urinary chloride concentration, which were statistically significant for females. It is possible that this was an anomalous finding as the usual effect of fluoride containing compounds is polyuria. It is possible that the reduced urine output during the overnight collection period was consequence of increased urine output during the exposure period.

A dose-related increase in urinary fluoride was evident in both sexes of all treated groups following 4 weeks of exposure and attained statistical significance for groups 3 and 4. Urinary fluoride levels for rats of group 4 remained higher than controls following 2 weeks of recovery and remained statistically significant. The presence of fluoride ion in urine is commonly associated with the metabolic breakdown of fluoride-containing compounds such as the test substance.

Kidney and liver weights for test groups were greater than controls following 4 weeks of exposure and attained statistical significance for groups 3 and 4. Kidney weights of females from group 4 remained higher than controls following 2 weeks of recovery. The changes in kidney weight were considered to be treatment-related and potentially associated with changes in renal function. In the absence of microscopic findings, it was considered that the increase in liver weight was of no toxicological importance.

The macroscopic examinations performed at termination revealed no treatment-related abnormalities. At the end of the exposure period, the microscopic examinations revealed treatment-related increased incidence of pseudogland formation in the respiratory epithelium in the nasal turbinates of males from group 4 and increased incidences of cortical tubules with hyaline droplets in the kidneys of males from all treated groups. At the end of the recovery period, the microscopic examinations revealed recovery to control level for pseudogland formation in the nasal turbinates of the respiratory epithelium. Almost complete recovery had occurred for cortical tubules with hyaline droplets of the kidney. The presence of hyaline droplets in cortical tubules in the kidneys of male rats is a common non-specific response to a range of chemicals and is considered to be due to exacerbated accumulation of alpha-2µ-globulin in the kidneys and probably of no significance to man.

At 15 and 51 ppm the adverse effects included microscopic changes in the nasal turbinates of minor significance and a reduced weight gain. Partial or complete recovery from all the effects seen in this study was considered to have been demonstrated. In this study the no-observed-adverse-effect-level, for male and female rats, was considered to be 5 ppm (i.e. 33 mg/m3).